Genetic engineering to delete enzyme genes

The use of genetically engineered overexpression vectors does not prevent non-target enzymes from also being produced by a microorganism. Ideally a production microorganism maximizes the expression of the enzyme of interest while minimizing production of inconsequential or deleterious enzymes such as proteases or enzymes with interfering activities. Deletion of genes coding for undesirable enzymes can be performed when necessary to prevent their production. T. reesei is a filamentous fungus...

Purification decolorization and finishing

The main 'purity' consideration for feed enzymes is not the removal of proteinaceous impurities, but rather ensuring that only food-grade raw materials and non-pathogenic, non-toxigenic production organisms are used. In some cases, an undesirable side activity must be removed through some means of purification, particularly when it is not feasible to delete the side activity genetically. Even if minor proteases co-secreted by the host organism do not cause significant degradation of the product...

Natural isolate screening

The source material can be plant or animal matter, or microbes both prokaryotes (e.g. bacteria and archaea) and eukaryotes (e.g. yeasts and fungi). Currently, microorganisms are the major source for industrial enzymes in the feed sector. The classical method of screening natural microbial isolates is a well established process having its roots in antibiotic discovery (e.g. penicillins, streptomycin) in the decades after 1945. It involves the examination of thousands of samples of soil, plant...

Amylase engineering

A similar strategy has been successfully employed with amylase. The a-amylase from B. licheniformis consists of 458 amino acid residues, which in three dimensions consist of three domains as shown in Fig. 13.3. The active site and substrate binding region are situated between domains A and B. The engineering goal for amylase was to increase the stability of the enzyme. Numerous variants with increased stability were found by exploiting the diversity found in related a-amylases from different...

Identification and Development of Novel Enzymes Enzyme identification

The development of enzyme products often relies on screening a large number of organisms for an enzyme activity with a specific set of biochemical and physical characteristics that suit the targeted application. Traditionally, microbial screening processes have involved evaluation of purified enzymes isolated from (i) pure cultures obtained from in-house or accessible culture collections throughout the world (ii) microorganisms isolated from environments rich in the substrate of interest and...

FaeA expression vector

A. niger expression vector for ferulic acid esterase A (faeA). The vector shows required elements to produce high level faeA expression a strong glucoamylase (glaA) promoter, the expressed gene, faeA and a selectable marker, pyrG. order to grow, cells must take up the overexpression vector DNA containing a pyrG gene. Those cells that take up the vector can synthesize pyrimidine and DNA.

Introduction

Enzymes were discovered in the latter part of the 19 th century and have been used in industrial and food processes since the early 1900s. Examples include the use of amylases in textiles for removal of the starch sizing, bovine rennin in cheese making, pancreatic enzymes in production of leather and degumming of raw silk, and proteases for beer stabilization. As a result of the advances in biotechnology over the past 10 20 years, especially in the areas of genetics and protein engineering,...

Subtilisin engineering

Enzymes are synthesized as linear polypeptide chains. The amino acid sequence is dictated by the DNA sequence of the structural gene. After being synthesized, the enzyme folds into a stable three-dimensional structure that brings residues far removed in the linear sequence into close juxtaposition. The juxtaposition of these residues and the creation of a substrate binding surface confer the enzyme molecule with its catalytic properties. As an example, the structure of subtilisin BPN', a serine...

Downstream Processing and Formulation of Enzymes

The overall goal of downstream processing and formulation is to recover the enzyme of interest cost effectively at sufficiently high yield, purity and concentration, and in a form that is stable, safe and easy to use in a target application. The variety of separations technologies available is large and the possible order and permutation of sequential processing steps is vast. Several general considerations, however, limit the range of practical choices for a process design Becker, 1995 ....

Harvest and cell separation

In general, cells should be removed from a fermentation broth within hours after harvest in order to prevent cell lysis. After cell separation, the clarified fermentation broth is usually stable and can be stored refrigerated for days. Upon harvest, the broth may need to be cooled, pH adjusted, and certain stabilizers added in order to minimize enzyme degradation. For the more labile enzymes such as proteases, control of temperature, pH, oxidants, inhibitors and activators will be important...

Classical mutagenesis

Classical mutagenesis is one of the most powerful techniques used to increase enzyme yields from microorganisms. Dramatic productivity improvements in the order of tenfold, or even 100-fold, are common. Classical mutagenesis techniques were originally developed by the pharmaceutical industry to improve the yields of antibiotics. The same techniques are used to improve enzyme yields. Classical mutagenesis techniques are not unlike the process of evolution. Both require mutations in the DNA of an...