酿酒酵母表面展示表达系统及应用

酵母细胞表面展示表达系统是一种固定化表达异源蛋白质的真核展示系统,即把异源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞,利用酿酒酵母细胞内蛋白转运到膜表面的机制（GPI锚定）使靶蛋白定位于酵母细胞表面并进行表达。它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白,可应用于生物催化剂、细胞吸附剂、活疫苗、环境治理、蛋白质文库筛选、高亲和抗体、生物传感器、抗原／抗体库构建、免疫检测及亲和纯化、癌症诊断等领域。国内对这一方面研究较少,本文主要介绍了该技术的基本原理、研究现状、应用及其发展前景。     

D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity

For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.     

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.     

酿酒酵母细胞表面工程应用研究新进展

酿酒酵母表面展示工程是一个新兴的蛋白表达系统,由于它能进行蛋白翻译后修饰,能方便地对表达的蛋白产物进行检测和筛选,近年来应用研究发展迅猛。它在构建全细胞催化剂、抗原/抗体库、生物吸附剂、生物传感器、组合蛋白文库、免疫检测及亲和纯化中取得了很多新的应用,在蛋白质分子的功能研究与应用中发挥了更加重要的作用。     

Comparison of the emulsion characteristics of Rhodococcus erythropolis and Escherichia coli SOXC-5 cells expressing biodesulfurization genes

Biodesulfurization of fuel oils is a two-phase (oil/water) process which may offer an interesting alternative to conventional hydrodesulfurization due to the mild operating conditions and reaction specificity afforded by the biocatalyst. For biodesulfurization to realize commercial success, a variety of process considerations must be addressed including reaction rate, emulsion formation and breakage, biocatalyst recovery, and both gas and liquid mass transport. This study evaluates emulsion formation and breakage using two biocatalysts with differing hydrophobic characteristics. A Gram-positive (Rhodococcus erythropolis) biocatalyst, expressing the complete 4S desulfurization pathway, and a Gram-negative biocatalyst (Escherichia coli), expressing only the gene for conversion of dibenzothiophene (DBT) to DBT sulfone, are compared relative to their ability to convert DBT and the ease of phase separation as well as biocatalyst recovery following desulfurization.     

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

A systematic and powerful knowledge‐based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case‐by‐case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole‐cell biocatalysts. They are still largely missing for whole‐cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole‐cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole‐cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.     

ENHANCEMENT OF PROPYL GALLATE YIELD IN NONAQUEOUS MEDIUM USING NOVEL CELL-ASSOCIATED TANNASE OF Bacillus massiliensis

Enzymatic synthesis of propyl gallate in organic solvent was studied using cell-associated tannase (EC 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as the biocatalyst. The effects of solvent, surfactant treatment, and bioimprinting on the propyl gallate synthesis were studied and subsequently optimized. Among various solvents, benzene followed by hexane was found to be the most favorable. Treatment of the biocatalyst with Triton X-100 at a lower concentration (0.2% w/v), before lyophilization, increased the propyl gallate yield by 24.5% compared to the untreated biocatalyst. The biocatalyst was imprinted with various concentrations of gallic acid and tannic acid. Biocatalyst imprinted with tannic acid showed 50% enhancement in the propyl gallate yield compared to the non-imprinted biocatalyst.     

An amplified mass piezoelectric immunosensor for Schistosoma japonicum

An ultrasensitive piezoelectric immunosensor using an amplification path based on an insoluble biocatalyzed precipitation product has proposed for Schistosoma japonicum. A mercapto Schistosoma japonicum antigen was self-assembled onto the quartz crystal surface via an Au nanoparticle mediator monolayer to sense the Schistosoma japonicum antibody (SjAb). And the horseradish peroxidase labeled protein A conjugate which was bounded to the SjAb by a "sandwich" format was used as a biocatalyst for the oxidative precipitation of 4-chloro-1-naphthol by H(2)O(2) to yield the insoluble product benzo-4-chlorohexadienone, resulting in an amplified mass sensing of antigen-antibody interaction. The amount of the precipitate accumulated on the quartz crystal is controlled by the antibody concentration. The SjAb can be linearly determined in the range of 10-200 ngml(-1) and the detection limit reaches as low as 5 ngml(-1).     

An extension of the pressure-test technique for immobilized biocatalyst preparations

Based on an analysis of variables in the Ergun equation for pressure drop in granular beds, the technique of pressuretesting of immobilized biocatalyst preparations is reexamined from the point of view of evaluation of biocatalyst mechanical properties in packed bed reactors. The variation of biocatalyst particle size in the course of bioreactor performance was found to be a convenient estimate of mechanical strength for the biocatalyst particles. Based on this finding, an extension of the pressure-test technique with particle size analysis was proposed as an opportunity to carry out differential analysis of biocatalyst structure, regardless of time and position. An example of bed destruction is given to support the suggested characterization analysis.     

Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation

A biocatalyst was prepared by immobilizing a commercial Saccharomyces cerevisiae strain (baker's yeast) on orange peel pieces for use in alcoholic fermentation and for fermented food applications. Cell immobilization was shown by electron microscopy and by the efficiency of the immobilized biocatalyst for alcoholic fermentation of various carbohydrate substrates (glucose, molasses, raisin extracts) and at various temperatures (30-15 degrees C). Fermentation times in all cases were low (5-15 h) and ethanol productivities were high (av. 150.6 g/ld) showing good operational stability of the biocatalyst and suitability for commercial applications. Reasonable amounts of volatile by-products were produced at all the temperatures studied, revealing potential application of the proposed biocatalyst in fermented food applications, to improve productivities and quality.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Kinetic aspects of glucose oxidation by Gluconobacter oxydans cells immobilized in calcium alginate

Summary Gluconobacter oxydans subspecies suboxydans (ATCC 621 H), when growing at high glucose concentrations, oxidizes this substrate incompletely and gluconic acid accumulates in the medium in almost stoichiometric amounts. Such cells were harvested and entrapped in various alginate gels. The preparation with the highest retention of glucose oxidizing activity was used in further studies with the aim of developing an efficient process for continuous gluconic acid production.The retention of activity increases (up to 95%) as the alginate concentration in the gel decreases or the cell/alginate weight ratio is enhanced. In the latter case, however, transport of oxygen to and inside the biocatalyst beads rapidly becomes rate-limiting and thus lowers the efficiency of the biocatalyst. Similarly, the efficiency decreases as the size of the biocatalyst beads increases. In no case rate-limitation by transport of glucose was found. Thus, biocatalyst activity per unit volume of support, diameter of the biocatalyst beads, and aeration efficiency are important parameters for reactor design.     

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.     

Stability of biocatalysts on the basis of carrageenan-immobilized Escherichia coli during continuous synthesis of L-malic acid

Continuous enzymatic synthesis of L-malic acid from potassium fumarate in packed-bed flow reactors was investigated. Carrageenan-immobilized Escherichia coli cells were used as a biocatalyst. The operational stability of the biocatalyst fumarase activity was studied, and conditions for preserving high activity of the biocatalyst were determined.     

Selection of a whole-cell biocatalyst for methyl parathion biodegradation

Whole-cell biocatalyst has the potential to become a cost-effective alternative to conventional enzyme methods for solving ecological and energy issues. However, cytosolic-expressing biocatalyst systems are critically disadvantaged due to the low permeability of the cell membrane. To overcome substrate transport barrier, periplasmic secretion and surface display biocatalysts were developed by expressing signal peptides or anchor proteins in Escherichia coli. In this work, six carriers were compared in regard to whole-cell activity of methyl parathion hydrolase (MPH). Our results indicate that the surface display systems yielded one to three times whole-cell activity than the periplasmic secretion systems. Although periplasmic secretion systems showed generally more stable than surface display systems, surface display appeared more suitable for whole-cell biocatalyst. It should note that the applicability of the DsbA/PhoA/AIDA-I leader to MPH expression is shown here for the first time. In addition, the result provided a useful reference for other whole-cell biocatalyst selection.     

Enhancement of phase separation by the addition of de-emulsifiers to three-phase (diesel oil/biocatalyst/aqueous phase) emulsion in diesel biodesulfurization

Ethanol, added as a de-emulsifier to separate oil and biocatalyst (or bacterial cells) from a three-phase (oil/biocatalyst/aqueous phase) emulsion, formed in diesel biodesulfurization employing Gordonia nitida, improved oil recovery by centrifugation from about 50% in its absence to almost 100% at 3% (v/v). The biocatalyst recovered with ethanol addition showed similar specific growth rates (0.03 h^–1) and dibenzothiophene desulfurization rates (6–7.2 mol l^–1 h^–1) to those (0.03 h^–1 and 7.1 mol l^–1, respectively) of the biocatalyst recovered with no ethanol addition. The desulfurization activity significantly increased as the number of the repeated recovery and reuse of the biocatalyst.     

Immobilization of mycobacterial cells onto silicone--assessing the feasibility of the immobilized biocatalyst in the production of androstenedione from sitosterol

Silicone rubbers are hydrophobic, a feature that may prove advantageous if this material is to be used as immobilization matrix in bioconversion systems where hydrophobic species are present, such as sterols and mycobacterial cells. Mycobacterium sp. cells with sitosterol side chain cleavage activity were accordingly effectively adsorbed onto silicone and the potential application of the concept was assessed by matching the behavior of the resulting immobilized biocatalyst with free cells and Celite immobilized cells. Mass transfer, kinetics, thermal and storage stability characterization of a biotransformation system based in the use of the silicone immobilized biocatalyst was performed. The feasibility of biocatalyst reutilization was tentatively explored.     

One-pot bio-synthesis of propyl gallate by a novel whole-cell biocatalyst

Propyl gallate has an excellent antioxidative capacity and some pharmaceutical potentials. In order to examine the feasibility for one-pot bio-synthesis of propyl gallate catalyzed by a whole-cell biocatalyst in organic media, a whole-cell biocatalyst of Aspergillus niger was prepared and utilized to catalyze the transesterification with tannic acid as a raw material. Furthermore, both the catalytic system and the reaction mode were optimized to further improve the conversion rate of substrate. The result shows that a promising conversion rate, 43%, was achieved by the pH-tuned mycelium-bound tannase. The rate is over than or very close to that achieved by isolated tannase. The study on reaction mode indicates that the simulated continuous catalysis is the most suitable to the transesterification as compared to batch catalysis and batch catalysis coupled with product separation. Accordingly, the one-pot bio-synthesis of propyl gallate by the novel whole-cell biocatalyst has such three advantages as easy operability of the biocatalyst, high efficiency of reaction mode, and the abundance of the natural raw material, which will contribute to constructing an efficient and eco-friendly method for one-pot synthesis of propyl gallate in an economical and ecological manner.     

Preparation of L-aspartic acid by means of immobilized Alcaligenes metalcaligenes cells

Two types of biocatalysts based on immobilized cells of Alcaligenes metalcaligenes exhibiting aspartate ammonia-lyase activity (EC 4.3.1.1) were developed for the enzymic preparation of L-aspartic acid from ammonium fumarate. The first type of the biocatalyst consists in individual covalently crosslinked and permeabilized cells(I), while the second type is represented by cell aggregates (II). For the above preparation, biocatalyst I can be used only discontinuously in a mixed reactor. After termination of the reaction between individual cycles of its use, the biocatalyst is returned to the reactor in the form of a highly concentrated cell suspension or paste. Biocatalyst II can be used discontinuously or continuously in a fixed-bed column of the catalyst. The effects of pH, substrate concentration and temperature on the reaction velocity and effectivity of enzymic conversion was investigated. Optimal parameters of the reaction are as follows: pH 8.5, initial substrate concentration, 1.35 mol/L, temperature for discontinuous process, 37 degrees C, and temperature for continuous process, 25 degrees C. Under these conditions the enzymic conversion of substrate to product is quantitative. Under optimal toring conditions, the specific activity of both catalysts does not change within a period of one year. The operational half-life of the biocatalyst II during continuous use in a fixed-bed column of the catalyst under standard reaction conditions depends on the quality of the substrate. The discontinuous preparation of L-asparatic acid with the aid of biocatalyst I and continuous preparation of this product with the aid of biocatalyst II have been verified under pilot-plant conditions.     

Enhancing the synthetic utility of aldolase antibody 38C2

Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzaldehyde and acetone to give 4-(4'-nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzaldehyde, the untreated 38C2 gave only 24% transformation in 18 h at 25 degrees C. However, since TPP-treated 38C2 also gave an additional (unidentified) product, its synthetic utility was limited. Crosslinked aggregate of 38C2, however, gave the biocatalyst which gave a single product and could be reused at 40 degrees C five times without loss of activity.